Gangliosides, glycosphingolipids containing sialic acid, are normal constituents of all cell membranes in mammals and are abundant in the nerve tissue (Ando S.: Neurochem. Int. 5:507, 1983). Four gangliosides, GM.sub.1, GD.sub.1a, GD.sub.1b and GT.sub.1b (nomenclature according to Svennerholm L., J. Neurochem, 10:613, 1963), constitute 80-90% of the total ganglioside content of the mammal brain. Gangliosides are specifically localized in the outer layer of the plasma membrane, suggesting that they play an important role in many biological activities, for instance as a "sensor" and/or receptor for various molecules, and in the transfer of information through the cell membranes (Fishman et al.: Science 194:906, 1976). They therefore play a key role in the regulation of neuronal development and repair in the central and peripheral nervous systems.
There is indeed ample documentation that gangliosides are able to favorably influence functional recovery following lesion in the peripheral nervous system (PNS) and central nervous system (CNS), by the involvement of specific membrane mechanisms and by interaction with neurotrophic factors as revealed by in vitro studies on neuronal cultures (Doherty P. et al., J. Neurochem. 44:1259, 1985; Skaper S. et al., Molecular Neurobiology, 3:173, 1989).
In particular, it has been reported that the administration of gangliosides in vivo facilitates nerve regeneration and functional recovery in the PNS under pathological conditions: positive effects have been described in models of traumatic neuropathies (Ceccarelli B. et al., Adv. Exp. Med. Biol. 71:275, Plenum Press, New York, 1976; Gorio A. et al., Brain Res. 7:236, 1980; Gario A. et al., Neuroscience 8:417, 1983), metabolic neuropathies (Norido F. et al., Exp. Neurol. 83:221, 1984) and toxic neuropathies (Di Gregorio F. et al., Cancer Chemother., Pharmacol. 26:31, 1990).
With regard to the CNS, positive effects have been widely reported of recovery induced by monosialoganglioside GM.sub.1 in models of ischemia (Cuello A. C. et al., Brain Res. 376:373, 1986; Karpiak S. E. et al., CRC Critical Rev. in Neurobiology, Vol. 5, Issue 3, 1990), traumatic lesion (Toffano G. et al., Brain Res. 296:233, 1984) and neuronotoxic lesion (Johnsson J., Dev. Brain Res., 16:171, 1984) in various neuronal systems of different animal species. It has recently been discovered that gangliosides can inhibit the translocation and activation of protein kinase C induced by glutamate (Vaccarino F. et al., Proc. Nat. Acad. Sci. USA, 84:8707, 1987). This action is very important in conditions of ischemic damage, where there have been reports of a crucial role played by excitatory amino acids, such as glutamate, which trigger a cascade of events leading to neuronal death. This mechanism could favor the survival of neurons in the area around the lesion, prevent retrograde degeneration, and accelerate the reparative growth response to local trophic factors.
The results of experimental research have been amply confirmed by those from the clinical use of gangliosides. For over ten years gangliosides have been used as therapeutic agents in almost all forms of peripheral neuropathy, from those forms resulting from mechanical damage to those caused by toxic factors or deficiencies, from infectious and inflammatory disorders to metabolic dysfunctions. These drugs have proved to be equally efficacious in mono and polyneuropathies, in sensory-motor disorders and in pathologies affecting the autonomic nervous system, such as in many neuropathies affecting the cranial nerves, for instance Bell's palsy, trigeminal neuralgia, and neuralgia caused by herpes zoster. Gangliosides, and in particular the monosialoganglioside, can be widely used in all pathologies connected with acute lesions in the CNS of a vascular or traumatic type and in the sequelae of such pathologies (cerebral ischemia, cranial and spinal trauma).
Their proven reparative activity in the CNS also supports their use in chronic neurodegenerative pathologies, such as Parkinson's disease and Alzheimer's disease. The fact that they are "endocoids" (endogenous drugs) by nature, being natural components of the neuronal membranes, explains their excellent tolerability and the absence, even in prolonged treatments with high doses, of side effects which are so frequent in some conventional therapies for peripheral neuropathies.
In general, suitable ganglioside mixtures, for example a formulation of the following kind: GM.sub.1 from 18% to 24%, GD.sub.1a from 36% to 44%, GD.sub.1b from 12% to 18%, GT.sub.1b from 16% to 22%, or the single ganglioside fractions, particularly the monosialoganglioside GM.sub.1, present biological activities such as those described. These gangliosides, as suitable mixtures or single fractions, in particular the monosialoganglioside GM.sub.1, are extracted from mammal brains and it is therefore necessary, given their particular biological function and their therapeutic application previously described with regard to the peripheral and central nervous systems, to utilize purification methods which guarantee a final product which is absolutely pure and free from biological and chemical contaminants.
It has long been known that it is possible to extract, on a research level, mixtures of gangliosides (Tettamanti et al., Biochim. e Biophys. Acta, 296:160, 1973; Trams et al., Biochim. e Biophys. Acta, 60:350, 1962: Bogoch et al., British J. Pharm., 18:625, 1962; Wiegandt et al., Angew Chem. 80:89, 1968; U.S. Pat. No. 3,436,413; and C. A. 61, 9851C, 9895d), but none of the aforesaid methods was developed with a view to demonstrating the elimination and destruction of components associated with non-conventional viruses. One reason for this is that, at the time, such diseases, affecting the mammalian species to which the brains used for extraction belonged, were as yet unknown. Another reason is that no reagents were available for the specific identification of potentially dangerous components, whereas today such reagents have been made available by specific methodologies developed on the basis of newly-acquired knowledge gleaned from the scientific evolution of molecular biology techniques.
Sometimes situations of a pathological type can arise wherein the pathogenic agent or agents cannot be identified. One such pathological situation is called bovine spongiform encephalopathy (BSE), first reported in England in 1986 (Wells G. et al., Vet. Record, 419, 1986). This name derives from the spongy appearance of the brain tissue from afflicted animals. When sections of tissue are analyzed by microscope, the main lesions are comprised by extensive neuronal vacuoles.
All available evidence points to the fact that BSE belongs to a group of degenerative encephalopathies of the central nervous system which are invariably fatal in outcome and are caused by a group of non-conventional, infectious agents (Fraser et al., Vet. Record 123:472, 1988; Hope et al., Nature 336:390, 1988). This group also includes scrapie of sheep and goats, the chronic emaciating disease which afflicts captive deer, infectious encephalopathy of mink on mink farms, and two human diseases; kuru and Creutzfeldt-Jacob disease. The histopathological lesions caused in the brain by these diseases are similar in all cases and are comparable to those caused by BSE. Many theories have been put forward on the nature of these etiological agents, which are neither bacteria nor virus, are unlike any other known organism and are therefore known as unconventional viruses. On account of their long incubation periods, running from the moment of infection to the onset of symptoms, these viruses are also known as "slow viruses".
Since the few cases observed in 1986, the disease spread and has reached epidemic proportions in Britain, affecting some 14,000 cattle and increasing steadily by about 250-300 cases each week. The infected cattle show no signs of disease for several years (the incubation period being 4-5 years), but once symptoms have appeared the animals rapidly deteriorate and die.
An epidemiological study by the Central Veterinary Laboratory of the British Ministry of Agriculture (Wilesmith et al., Vet. Record. 123:638, 1988) showed the source of infection to be animal fodder made with the processed carcasses of other ruminants, sold in the form of powdered meat or bone. Since the encephalopathy can be transmitted to a wide range of animal species, it seems reasonable to assume that BSE is the result of infection by the etiological agent responsible for scrapie, transmitted from sheep by means of these contaminated foodstuffs (Morgan K L, Vet. Record 122:445, 1988).
On the basis of the results of this study, the British government banned, by an order which came into force on 18th Jul. 1988, the sale and supply of animal foodstuffs containing animal proteins derived from ruminants.
The general opinion is that many factors have contributed together to the sudden appearance of BSE in Britain (Cherfas J., Science, Feb. 1990, 523).
Firstly, the number of sheep in Great Britain increased rapidly in the late 70's and early 80's, and with this the incidence of scrapie, an endemic disease of sheep in Europe for over 250 years (Pattison et al., Vet. Record 90:465, 1972). At the same time, in the wake of the petrol crisis, factories producing animal fodder changed their methods of processing carcasses to a lower-temperature system which was probably less efficient in destroying the highly resistant scrapie agent. All except one of the producers of these foodstuffs abandoned the use of solvents such as benzene, hexane and trichloroethylene, to remove excess fats from soybean and bone meal. Perhaps most significant of all was that the final stage of heating of the products to remove the solvents was consequently left out: indeed this phase required very high temperatures.
Moreover, government policy encouraged breeders to produce more milk, and wean calves early by feeding them protein-rich diets. These animals were often of poor quality, since meal made from meat and bone was cheaper than products made with soybean and fish which are surer sources of protein. Studies to find how the disease is transmitted are fundamental to BSE research. The most important aspect of these experiments is that, by identifying the limits of the inter-species barriers to transmission of the pathogenic agent, it is possible to assess the risk of BSE infection to any one species. Fraser et al. (Vet. Record, 123:472, 1988) demonstrated that the disease could be passed from cattle to mice. They inoculated extracts from the brains of cattle which had died from BSE into the brains of mice which subsequently developed the disease. Later, Barlow et al. (Vet. Record, 3 Feb. 1990) transmitted the disease to mice by feeding them infected brains. It was the first proof that BSE could be contracted by eating infected material. No other tissue from afflicted animals (spleen, spinal cord, lymphatic tissues, milk etc.) was able to produce the disease in mice.
There is proof that scrapie can be transmitted to lambs by their mothers, but so far no evidence has come to light of possible vertical or horizontal transmission of the etiological agent of BSE in cattle.
The agents which cause subacute infectious encephalopathies are extremely resistant to standard decontamination processes. Available data on this aspect mostly originate from studies on the inactivation of agents of scrapie and Creutzfeldt-Jacob disease. The etiological agent of scrapie is highly resistant to temperature change. When exposed to temperatures of up to 80.degree. C. their infectiousness is only slightly reduced; higher temperatures however markedly reduce infectiousness (Hunter et al., J. Gen. Microbiol. 37:251, 1964). A small quantity of infectious "virus" sometimes persists when suspensions of infected material are heated to 100.degree. C. for 1 hour or to 118.degree. C. for 10 minutes.
Recently, the need was felt to renew standards of sterilizing these infectious agents under high steam pressure in autoclaves. The current standards governing autoclaving in the United States for the decontamination of Creutzfeldt-Jacob disease involve treatment at 132.degree. C. for 1 hour (Rosenberg et al., Annals of Neurology 19:75, 1986), and is based on studies carried out on brain homogenates containing scrapie or Creutzfeldt-Jacob agents (Brown et al., J. of Infectious Diseases 153:1145, 1986). In Britain the current standard of autoclaving for decontamination from Creutzfeldt-Jacob disease involves treatment in an autoclave at 134.degree.-138.degree. C. for 18 minutes, on the basis of some studies including one by Kimberlin (Kimberlin et al., Journal of Neurological Sciences 59:355, 1983). Unfortunately, the bovine spongiform encephalopathy agents are very resistant even to common chemical treatments, as well as physical ones. Solvents such as benzene, hexane, petrol and trichloroethylene have been used as extraction solvents, but little is known of their effects on infectivity. Only a small quantity of data is available on the chemical inactivation of infective agents, mainly because studies require large numbers of animals and long observation times. Concentrations of 0.3%-2.5% of sodium hypochlorite greatly reduced infectivity in the biological assays used, but often did not completely eliminate it (Walker et al., Am. J. Publ. Health 73:661, 1983). Data regarding treatment with up to 0.25N sodium hydroxide are very variable; at concentrations of over 1N it appears however to be the most efficacious chemical agent of all those studied. Treatment with 6M-8M urea was also reported to be highly variable.
The results of the studies on decontamination thus show that, although most of the infectivity is quickly destroyed by many of the different physical and chemical processes, the existence of small subpopulations of resistant infectious agents makes sterilization of contaminated materials extremely difficult in practice.
Once BSE had been identified as a "scrapie-like" disease, important questions began to be asked on epidemiological and analytical levels, the latter in particular being aimed at identifying the agent associable with infectivity. However, all efforts so far made to identify nucleic acids associated with the etiological agent have been unfruitful. The only component isolated, which is associated unequivocally with the infective action, is a sialoglycoprotein called scrapie prion protein (Prp.sup.Sc).
Genetic studies conducted on this protein subsequently provided some surprising information. Some DNA probes synthesized according to the N terminal sequence of the protein have made it possible to show the presence of a chromosome gene in individual copy that exhibits the same restriction pattern both in the brains of healthy animals as in the brains of infected animals. This gene, which is conserved even in very different species, codes a protein called cellular prion protein (PrP.sup.C) with an apparent molecular weight of 33-35.0 kilodaltons (kd), which shows particularly evident differences with respect to the PrP.sup.Sc :
1) PrP.sup.C is susceptible to protease, while PrP.sup.Sc is resistant. In particular, while PrP.sup.C is degraded completely by the enzyme proteinase K, PrP.sup.Sc is hydrolyzed at the level of the N terminal for a fragment of about 5 kd and gives rise to a protein called PrP.sub.27-30. This form copurifies with the infectivity and is the most abundant component that is obtained in the preparations of infective material.
2) Both PrP.sup.C and PrP.sup.Sc are membrane proteins, but while the first is solubilized by treatment with detergents, the second tends to polymerize into amyloid fibrous structures. Similar structures (scrapie-associated fibrils, SAF) have been found in infected brains and are peculiar for this type of infection. The resistance of this infective protein to inactivation is unusual: it is sensitive, for example, to treatments with concentrated alkaline solutions or to exposure to temperatures above 120.degree. C. and to their combinations or combinations with different denaturing agents. Consequently, the only diagnostic methods available for unequivocal identification of these spongiform encephalopathies are the verification of the presence of the SAF in the infected cerebral tissues, extraction and immunochemical identification of the protein PrP.sub.27-30, methodologies applicable only during pathological anatomy.
The SAF have been identified on infected bovine brains, then the homolog of the PrP.sup.Sc was isolated and showed reactivity with a serum obtained against mouse SAF. Further, the N terminal sequence of the first 12 amino acids showed 100% homology with the PrP.sup.Sc of sheep and a difference from that of the mouse, hamster and man by a single insertion of glycine. As soon as it was established that BSE is a "scrapie-like" disease, some important questions arose at the epidemiological and analytic level, the latter particularly devoted to identifying the protein associable with infectivity.
The unexpected cropping up of BSE and all the aspects still to be explained on these neurological disorders have caused a necessary consideration to be given to the problem, especially by those involved in the preparation of products that derive from bovine material.
It could, in fact, not be enough to use, for obtaining compounds or their mixtures of pharmaceutical interest, raw material certified for food use. Consequently, it is necessary to develop the process of production of the products in question by using extraction methodologies that guarantee the elimination of the protein associated with infectivity and the infectivity itself. It is obvious that the process of extraction of the infectively active fraction should, at the same time, preserve the biological activity of the active principles desired as final product.
The other potentially dangerous protein at the level of these preparations for man is the myelinic basic protein (MBP).
It is a protein that in man and most vertebrates has a molecular weight (MW) of about 19.5 kd. It is present in three molecular forms in human myelin and in six in that of the mouse, coding by a single gene located on chromosome 18 and it constitutes about 30% of the total myelinic protein. Its exact topographical location is not yet certain. It has been observed in the cytoplasm of oligodendrocytes only at the moment of myelinization. A protein considered identical is present in the peripheral nervous system (protein P1), and the ability of peripheral myelin to induce experimental allergic encephalomyelitis (EAE) in laboratory animals is due to it.
Not all the molecule of MBP is encephalitogenic but only a portion that varies from species to species: in the rabbit the encephalitogenic portion is amino acid fragment 64-73, in the Lewis rat 71-85, in the guinea pig 113-121, in the SJL/J mouse 89-169. The EAE is a typical cell-dependent autoimmune disease; indeed, it is transferable from one animal to another by infusion of the sensitized T lymphocytes and not by infusion of serum. In this case, the disease is supported by transfused lymphocytes and not those of the recipient. Another cell-dependent autoimmune form is allergic experimental neuritis (EAN). It too can be induced in all species of higher vertebrates by inoculation of crude homogenate of peripheral myelin in complete Freund's adjuvant. It is considered that the antigen mainly responsible for this autoimmunization is protein P2, with an MW of 12.0 kd, present in the peripheral nervous system.
Another contaminant to be considered in these preparations is bovine genomic DNA. The possibility of being able to produce, by recombinant DNA technology, biologically active proteins, which can be used as pharmaceutical agents, has made it necessary to analyze the final products for the presence of DNA residues, belonging to the cell where the desired protein is expressed. The presence of DNA fragments in pharmaceutical preparations to be used in man poses the problem of the danger of an incorporation in the genome of these fragments with a possible uncontrolled transfer of genetic type information. Even though it is not yet possible to obtain gangliosides by recombinant DNA technology, it is necessary to apply this type of control analytical technology to extraction products that use raw material of animal origin.
Finally, the gangliosides to be used in vitro and in vivo studies should be free from other compounds such as asialogangliosides and glycocerebrosides. These substances, if present in high concentrations, can have important immunological implications and can also lead to erroneous experimental considerations.
The sudden onset of BSE and all the other aspects still to be clarified on these neurological disorders have caused necessary consideration to be given to the problem, especially by those involved in the preparation of products deriving from bovine material.
Earlier processes for preparation of gangliosides, such as cited above, required the product to be pharmaceutically acceptable, free from those biological contaminants which were known at the time to be potentially damaging to the health. But clearly, the subsequent onset of the aforesaid pathology in adult cattle has made it necessary to obtain an active principle which, without losing the aforesaid therapeutic properties, is characterized by the assured absence of non-conventional viral agents, to be achieved by the use of specific processes to guarantee the inactivation of these non-conventional viral agents and the complete elimination of infectivity, and to use specific methodologies by which to identify such agents. Indeed, it may not be enough to use raw material which has been certified as suitable for consumption, to obtain compounds or mixtures of the same for pharmaceutical purposes. Obviously, the onset of BSE must be assessed by taking into account its biological action in vivo, which must be considered as an example of verification of the various phases of the process, but not as a summary of the same. This analysis of the biological action in vivo is necessary since scientists are not yet in agreement over associating the infection with certain proteins such as PrP.sub.27-30. Clearly, the extraction process which eliminates infectious activity must at the same time leave the biological activity of the active principle intact, since this is essential for its therapeutic use. (Ad hoc working party on biotechnology/pharmacy: Validation of virus removal and inactivation process. Commission of the European Communities, March 1990). Scientific research has produced, on the one hand, methods which guarantee suitable mixtures of gangliosides or their single fractions to be obtained in forms free from protein, chemical and biological contaminants, and on the other hand, methods with demonstrated efficacy in destroying infectivity associated with slow viruses, but no method is known by which it is possible to obtain, also on an industrial scale, the similarly unknown result of a product, as desired, pure, pharmacologically active product, free from infectivity associable with pathogenic agents definable as slow viruses.